ES2641570T3 - Macromolecular delivery systems for non-invasive imaging, evaluation and treatment of arthritis and other inflammatory diseases - Google Patents

Abstract

A pharmaceutical composition for use in the treatment of an inflammatory disease comprising: a water-soluble N-2 (2-hydroxypropyl) methacrylamide (HPMA) copolymer and an effective amount of an anti-inflammatory therapeutic agent bound to said soluble HPMA copolymer in water, wherein the water-soluble HPMA copolymer is attached to the anti-inflammatory therapeutic agent through a spacer comprising a hydrazone bond.

Description

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DESCRIPTION

Macromolecular delivery systems for non-invasive imaging, evaluation and treatment of arthritis and other inflammatory diseases

Technical field of the invention

This invention relates to biotechnology, more particularly, to water soluble polymer delivery systems for non-invasive imaging, the evaluation and treatment of arthritis and other inflammatory diseases.

Background of the invention

Rheumatoid arthritis (RA) is the most common inflammatory arthritis, which affects approximately 1% of the general population worldwide. In the United States, about 4.5% of people over 55 have been affected (1, 2).

As a symmetric disease, RA usually involves the same joints on both sides of the body. Angiogenesis and microvascular lesions are common features of RA inflammation, which leads to an abnormal infiltration of the serum protein in the synovium (3-5). Damaged or reduced lymphatic cells have also been observed in the synovium of patients with RA (6, 7).

Although the exact cause of rheumatoid arthritis is unknown, many medications have been developed to relieve its symptoms and slow or stop its progression. The most commonly used drugs are based on three main approaches: symptomatic treatment with non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids and disease-modifying anti-rheumatic drugs (DMARDs) (3).

Considerable effort has been made to identify and develop new therapeutic strategies for the treatment of RA. Medications for aR, such as the specific cyclooxygenase-2 (COX-2) inhibitor (a FAINE) (8), tumor necrosis factor (TNF) blockers and interleukin 1 (IL) receptor antagonists -1Ra) (FARME) have been used for weather applications (3). Although the new generation of anti-rheumatic drugs has greater specificity for its molecular objective, the majority of them have no specificity for the diseased tissue, which leads to various side effects that limit their chemical application. The well-known side effects of FAINE include indigestion, stomach bleeding, liver and kidney damage, tinnitus buzzing, fluid retention and high blood pressure (9). Well-known side effects of corticosteroids include bruising, bone thinning, cataracts, weight gain, redistribution of fat, diabetes and high blood pressure (10). Some DMARDs are immunosuppressants and usually have serious side effects, such as increased susceptibility to infection (3). The recent withdrawal of Vioxx® (COX-2 inhibitor, Merck) is a good example of the tremendous impact that side effects can have on an otherwise effective drug.

The ubiquitous in vivo distribution of receptors used by the mayone of anti-rheumatic drugs is a leading cause of their side effects. Therapeutic administration systems, which could specifically provide arthritis drugs to the diseased tissue of patients with RA, can avoid many of the side effects manifested in other tissues while achieving much greater therapeutic therapeutic efficacy.

The application of water-soluble polymers as a drug vehicle for the effective administration of the drug to the desired sites (macromolecular therapy) has been extensively studied during the last two decades in the treatment of solid tumors (11). Due to the "permeable" vasculature and the underdeveloped lymphatic system, extravased macromolecules can accumulate efficiently in the solid tumor. This phenomenon is called "selective permeability and enhanced retention" (PRM) of the tumor and has been used successfully to direct cancer drugs against solid tumors (12).

D. Wang et al. (Inhibition of cathepsin K with lysosomotropic macromolecular inhibitors, Biochemistry, vol. 41, no. 28, July 16, 2002, pages 8849-8859) discloses conjugates of an HPMA copolymer for a cathepsin K inhibitor to treat osteoporosis and rheumatoid arthritis.

Studies using carriers in microparticles, such as liposomes for the supply of antiartic agents to an AR joint, indicate some promising results in an animal model of arthritis (13). However, liposome hepatotropism can be problematic due to secondary toxicity to the liver. Therefore, there is a need in the art for an effective drug delivery system intended for the appropriate tissues.

Description of the invention

The present invention is defined in the appended claims. Matters that are not covered by the scope of the claims are not part of the presently claimed invention. The invention relates to water soluble polymeric delivery systems. In one embodiment, the delivery system is used for the administration of drugs to the diseased sites for the treatment of rheumatoid arthritis and other inflammatory diseases. In other

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embodiment, the delivery system is used for the administration of imaging agents to the diseased sites for non-invasive imaging and the evaluation of the diseased sites of arthritis and other inflammatory diseases.

In one embodiment, the invention provides water-soluble delivery systems for the delivery of anti-inflammatory therapeutic agents selected from the group consisting of proteins, peptides, FAINE, FARME, glucocorticoids, methotrexate, sulfasalazine, chlorine, gold, gold salt, Copper, copper salt, penicillamine, D-penicillamine, cyclosporine, etc., and mixtures thereof, such drugs are well known to those skilled in the art (37, 38). Also disclosed herein is a water-soluble polymeric delivery system for the supply of imaging agents, which are useful for non-invasive imaging and for the evaluation of arthritis joints and other diseased organs or tissues. Imaging agents can be selected from any of the known compounds, for example, compounds useful for imaging by RM, PET, CT or gamma scintigraphy, etc. and mixtures thereof, such agents are well known to those skilled in the art.

In another exemplary embodiment, the invention provides a water soluble polymer delivery system for the administration of a combination of imaging agents and anti-inflammatory therapeutic agents. In another exemplary embodiment, the invention provides a water soluble polymer delivery system for use in a method of treating an inflammatory disease and monitoring the progress of treatment. In another exemplary embodiment, the invention provides a method of screening anti-inflammatory therapeutic agents, in which the anti-inflammatory agent is linked to a water-soluble polymer delivery system of the invention and administered to a subject, the effect is monitored. of the therapeutic agent, for example, using an imaging agent, and an effective therapeutic agent is identified. Optionally, an imaging agent can be co-administered in order to monitor and / or screen the activity of the anti-inflammatory agent. Optionally, a fraction or addressing fractions can be used in the screening method.

In another exemplary embodiment, the inflammatory disease is rheumatoid arthritis, osteoarthritis, temporomandibular joint syndrome, inflamed nerve root, Crohn's disease, chronic obstructive pulmonary disease, psoriatic diseases, asthma, colitis, multiple sclerosis, lupus erythematosus, atherosclerosis and /or similar. Also disclosed herein are drug delivery systems comprising a water soluble polymer main chain, optionally, a targeting fraction or fractions, and a therapeutic agent or agents and / or an imaging agent. The link (or links) between the addressing fraction (or fractions) and the main chain of the polymer is not degradable or degradable under physiological conditions. The bond (or bonds) between the therapeutic agent (or agents) and the polymer backbone is not degradable or degradable under physiological conditions. Also disclosed herein are delivery systems for imaging agents comprising a water soluble polymer main chain, optionally, a targeting fraction or fractions, and an imaging agent or agents. The link (or links) between the addressing fraction (or fractions) and the main polymer chain is not degradable or degradable under physiological conditions. The link (or links) between the imaging agent (or agents) and the polymer backbone is not degradable or degradable under physiological conditions. Also disclosed herein is a method of manufacturing a pharmaceutical composition and / or a medicament comprising one or more systems of administration of the invention for the treatment of rheumatoid arthritis, osteoarthritis, temporomandibular joint syndrome, inflamed nerve root disease, Crohn, chronic obstructive pulmonary disease, psoriasic diseases, asthma, colitis, multiple sclerosis, lupus erythematosus, atherosclerosis and / or the like.

As will be apparent to a person skilled in the art based on the invention described herein, the invention provides the advantage of incorporating multiple therapeutic agents, targeting fractions, bioassay markers, spacers and / or imaging agents that may include a plurality of different chemical species of one or more of these groups. Therefore, in yet another exemplary embodiment, the therapeutic agents, targeting fractions, bioassay markers, spacers and / or imaging agents may consist of any number or combination of different species, which have the same or different effects. .

Brief description of the drawings

Figure 1 illustrates the chemical structure of a polymeric delivery system for an MRI contrast agent (DOTA-Gd3 +), abbreviated as P-DOTA-Gd3 +, for the imaging of arthritis joints and disease severity assessment.

Figure 2 shows the histology of the ankle and knee joints of rats with adjuvant-induced arthritis (IAA) that were also visualized by MRI. Figure 2A is a low power micrograph of the ankle and foot bones. Extensive swelling and inflammation are evident in the soft tissues (*) that surround the bones of the foot. T = warm. Figure 2B shows the tarsal joint illustrating inflamed synovium (synovitis), extensive inflammatory infiltration (*) and destruction of cartilage and bones. B = bone, A = articular cartilage. Figure 2C is a micrograph of greater power of the inflamed synovium. A = articular cartilage. Figure 2D shows extensive bone destruction with inflammatory infiltration (*) in a tarsal bone (ankle). Bone surfaces are lined with large active osteoclasts (arrows). Figure 2E illustrates several blood vessels in an inflamed region of the ankle joint that illustrates the inflammatory reaction around the vessels. The endothelial lining is thickened and

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vacuolated (arrows). Figure 2F is a low power micrograph of the knee joint of this same animal. The joint is quite normal in appearance, except for a small pocket of inflammation in the posterior aspect of the joint (arrow). This same area was contrasted when observed by MRI. T = tibia; F = femur.

Figure 3 shows the MR images of the animals taken at different time points. The acquired images were subsequently processed using the maximum intensity projection algorithm (PMI). Figure 3A shows the rat base line with AIA; Figure 3B shows a rat with AIA, 5 minutes after the injection of P-DOTA-Gd3 +; Figure 3C shows a rat with AIA, 1 hour after the injection of P-DOTA-Gd3 +; Figure 3D shows a rat with AIA, 2 hours after the injection of P-DOTA-Gd3 +; Figure 3E shows a rat with AIA, 3 hours after the injection of P-DOTA-Gd3 +; Figure 3F shows a rat with AIA, 8 hours after the injection of P-DOTA-Gd3 +; Figure 3G shows a rat with AIA, 32 hours after the injection of P-DOTA-Gd3 +; Figure 3H shows a rat with AIA, 48 hours after the injection of P-DOTA-Gd3 +; Figure 3I. Healthy rat, base lmea; Figure 3J shows a healthy rat, 5 minutes after the injection of P-DOTA-Gd3 +; Figure 3K Healthy rat, 1 hour after the injection of P-DOTA-Gd3 +; Figure 3L shows a healthy rat, 2 hours after the injection of P-DOTA-Gd3 +; Figure 3M Healthy rat, 8 hours after the injection of P-DOTA-Gd3 +; Figure 3N shows healthy rat, 48 hours after the injection of P-DOTA-Gd3 +; Figure 3O shows a rat with AIA, 5 minutes after the injection of OM-NISCAN; Figure 3P shows a rat with AIA, 2 hours after the injection of OM-NISCAN; Figure 3Q shows a rat with AIA, 8 hours after the injection of OM-NISCAN; Figure 3R shows a rat with AIA, 32 hours after the injection of OM-NISCAN; Figure 3S shows a rat with AIA, 48 hours after the injection of OM-NISCAN; Figure 3T shows a healthy rat, 5 minutes after the injection of OM-NISCAN; Figure 3U shows a healthy rat, 1 hour after the OM-NISCAN injection; Figure 3V shows a healthy rat, 2 hours after the injection of OM-NISCAN; Figure 3W shows a healthy rat, 8 hours after the OM-NISCAN injection; Figure 3X shows a healthy rat, 48 hours after the OM-NISCAN injection.

Figure 4 illustrates a single-plane MR imaging of rats with IAA injected with P-DOTA-Gd3 +. Figure Fig. 4A shows an MR image of the base lmea of a rat with AIA; Figure 4B shows an MR image of the ankle and left leg of a rat with AIA, 2 hours after the injection; Figure 4C shows an MR image of the ankle and left leg of a rat with AIA, 8 hours after the injection; Figure 4D shows an MR image of the left knee joint of a rat with AIA, 8 hours after the injection.

Figure 5 illustrates the general structure of the water soluble polymer delivery system. The average mole percent of the addressing fractions (T) per polymer chain may vary from 0% to about 50%, preferably from 0% to 30%; the average mole percent of the therapeutic agents or imaging agents (D) or mixture of both by polymer chain can vary from 1% to about 90%; The average mole percentage of the bioassay marker (L) per polymer chain can vary between 0% and about 50%. The spacer S1 comprises a hydrazone joint. The spacer S2 may be of covalent or rtic bonds or bonds, such as peptides or other complex chemical structures, which may or may not be cleaved by stimulation, such as change of pH, specific enzymatic activity (for example, cathepsin K, MMP, etc. .), presence or absence of oxygen, etc., under physiological conditions. Spacer S3 illustrates a non-degradable covalent or physical bond or bond, under physiological conditions. The optional biodegradable cross-linking (C) can be covalent or physical bonds or bonds, such as peptides or other complex chemical structures, which can be cleaved by stimulation, such as a change in pH, specific enzymatic activity (e.g., cathepsin K, MMP, etc.), presence or absence of oxygen, etc., under physiological conditions.

Figure 6 illustrates the chemical structure of the polymeric drug delivery system by way of example, with dexamethasone as an example of a therapeutic agent, for the treatment of arthritis. The polymeric prodrug is abbreviated as P-Dex.

Figure 7 shows the superior therapeutic effect of P-Dex on dexamethasone sodium phosphate (Dex) in reducing the size of inflamed arthritis joints during treatment.

Figure 8 shows the superior therapeutic effect of P-Dex on dexamethasone sodium phosphate (Dex) in bone mineral density (BMD) of inflamed arthritis joints during treatment. The results were obtained by dual X-ray absorption (DEXA).

Figure 9 shows the superior therapeutic effect of P-Dex on dexamethasone sodium phosphate (Dex) in reducing the erosion of the bone surface of inflamed arthritis joints during treatment. The results were obtained by histomorphometna using a Bioquant image analysis system.

Figure 10 shows the superior therapeutic effect of P-Dex on dexamethasone sodium phosphate (Dex) by histological observation of inflamed arthritis joints during treatment.

Best mode or modes for carrying out the invention

Throughout the description of the invention and the claims, and following the convention, the "singular" includes the "plural"; for example, a therapeutic agent and / or a targeting fraction means at least one such agent

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therapeutic or addressing fractions, unless otherwise indicated.

To demonstrate the principle of the invention, conventional techniques of visual examination with Evans blue dye (EB) and magnetic resonance imaging (IMR) were used to follow the in vivo fate of macromolecules in a rat model established with AIA. In addition, the histological examination confirmed the presence of the disease in specific anatomical locations where the macromolecular delivery system is identified with the MRI technique.

EB is an agent commonly used to assess vascular permeability and integrity (23). It is a dye with multiple charges and aromatic structures, which forms a strong complex with plasma albumin. The dye injection has been used successfully to establish the concept of macromolecular therapy for the treatment of solid tumors (24). In this experiment, the EB dye technique was used in rats with AIA to visually assess the accumulation of plasma albumin in inflamed joints. The hind leg of the rats with AIA, where the most severe inflammation was evident, easily incorporated the dye compared to that observed in healthy rats. This observation confirmed that there was actually a much higher concentration of plasma albumine in the inflamed joints of the rat model with IAA.

Although the results with EB are significant, it is observed that the dye does not covalently bind to albumin and some transfers of dye not specifically to other tissues. For example, a slight blue spot was evident in some organs, including the Idgado and the heart.

Magnetic resonance imaging (MRI) is a non-invasive method of mapping the internal structure of the body. It uses radiofrequency (RF) radiation in the presence of carefully controlled magnetic fields in order to produce high quality cross-sectional images of the body in any plane. It represents the distribution of hydrogen nuclei and parameters related to their movement in water and lipids. The introduction of paramagnetic contrast agents shortens Ti (the longitudinal relaxation time) of the hydrogen nuclei in the tissues, which in turn will increase the intensity of the RM signal thereof (14). Therefore, to further support the results, magnetic resonance imaging (MRI) was used to track macromolecules marked with DOTA-Gd3 + injected in rats with AIA.

It is well understood that obtaining a higher intensity of the MR contrast signal in the MR images represents the existence of a higher concentration of paramagnetic contrast agents in the tissue. The analysis of the improved MR images of the macromolecular contrast agent of the rats provides important information on the pharmacokinetic profile and biodistribution of the water-soluble polymeric delivery systems described in this invention. In addition, such imaging agents will also increase the sensitivity and anatomical resolution of the images resulting from a subject (preferably a mairnfero, such as a human being), an animal (including an animal model for a particular disease, dog, cat, horse or cattle), or part (for example, a tissue or structure) of the subject or animal.

The conjugation of a paramagnetic contrast agent of low molecular weight, DOTA-Gd3 + complex with the HPMA copolymer allowed non-invasive monitoring of the fate of the injected polymer in rats with an rM scanner. This approach to labeling the polymer with an MRI contrast agent is similar to marking the polymers with fluorochromes to allow localization in organs, tissues and cells. Alternatively, this imaging approach can also be used with other imaging agents for PET, CT and gamma scintigraphy for non-invasive imaging purposes, evaluation of diseased tissues and organs and detection of molecular targets in the tissues or organs of interest, etc.

Therefore, a macromolecular magnetic resonance imaging (MRI) imaging contrast agent based on a copolymer of N- (2-hydroxypropyl) methacrylamide (HPMA) has been synthesized to illustrate the invention. After the systematic administration of the contrast agent in a rat model of adjuvant-induced arthritis (AIA), enhanced contrast MRI images were taken, showing the distribution of the polymer at different time points. The correlation of the MR results with additional visual and histopathological results of the rats with IAA, demonstrates the preferential deposition and retention of macromolecules in the inflamed joints. Therefore, the efficacy of the use of macromolecular therapy for the treatment of rheumatoid arthritis is demonstrated. In addition, these results demonstrate the feasibility of using macromolecular imaging agents for imaging and evaluation of artistic joints.

The invention includes polymeric delivery systems for the administration of anti-inflammatory drugs.

Polymeric delivery systems may further comprise an imaging agent for the delivery of imaging agents, such as chemical compounds used as enhancing agents in MRI (for example, DOTA-Gd3 +, DTPA-Gd, etc.), PET (for example , compounds labeled or complexed with 11C, 13N, 15O, 18F, 64Cu, 68Ga, 82Rb, etc., such as 18F-FDG), Ct (for example, a compound containing iodine or barium, such as acid 2,3, 5- triiodobenzoic) and gamma scintigraphy imaging (for example, compounds complexed with 99Tc, 111In, 113 In and 153Sm, etc.).

MRI procedure

The MR images of the animals were acquired in a 1.5 T Signa LX imaging system

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(General Electric Medical Systems, Milwaukee, WI), using a phase array coil. The images were acquired using an FSPGR sequence prepared for three-dimensional IR of a single slab in the coronal plane. The most common imaging parameters were TR = 13.4 ms, TE = 2.2 ms, TI = 300 ms, angle of rotation of 25 °, acquisition matrix in a 512 x 256 plane, field of vision (FOV) of 20 x 10 cm2, 64 portions per slab, 1.0 mm thick portions with 2X to 0.5 mm interpolation.

Poly synthesis (HPMA-co-APMA-co-MA-FITC).

HPMA (1 g, 7 mmol), APMA (0.14 g, 0.78 mmol), MA-FITC (0.043 g, 7.8 mmol), AIBN (0.057 g, 0.35 mmol) and MPA ( 0.001 mL, 1 mmol) in methanol (10 mL), were placed in a vial and purged with N2 for 5 minutes. The vial was sealed to the flame and kept at 50 ° C for 24 hours. The polymer was isolated by precipitation of the resulting solution in acetone and precipitated again twice. After drying the polymer in a desiccator (over NaOH), the final yield was determined as 0.9 g. The content of free amino groups in the copolymer was determined as 7.7 x 10 "4 mol / g using the ninhydrin test (18).

P-DOTA Synthesis

Poly (HPMA-co-APMA-co-MA-FITC) (170 mg, [NH2] = 1.33 x 10-4 mol), DOTA-NHS ester (100 mg, 2 x 10-4 mol) were mixed and diisopropylethylamine (DIPEA, 160 mL, 9.33 x 104 mol, distilled from ninhydrin) in DMF (1.5 mL, distilled from P2O5) and stirred overnight. The conjugate was precipitated in ether and dried in a vacuum. The product was further purified on an LH-20 column, dialyzed (the molecular weight cut-off size is 6-8 kDa) and lyophilized to obtain 190 mg of final product. The residue-free NH2 group was determined with a ninhydrin test and the DOTA content in the product was calculated as 7.5 x 10-4 mol / g.

Synthesis and purification of the macromolecular contrast agent IRM P-DOTA-Gd3 +

P-DOTA (100 mg, [DOTA] = 6.9 x 10-5 mol) and GdCh ^^ O (38 mg, 1.04 x 10-4 mol) were dissolved in 2 mL of deionized H2O. The pH of the solution was maintained at 5.0-5.5 overnight by gradual addition of NaOH solution (1 N). EDD disodium salt (38 mg, 1.04 x 10-4 mol) was then added to the solution to chelate the excess Gd3 +. After stirring for 30 minutes, the milky solution was purified with a Sefadex G-25 column to separate the Gd3 + chelated with EDTA and other low molecular weight compounds that did not react from the polymer conjugate. The conjugate was lyophilized to produce 115 mg of P-DOTA-Gd3 +. The gadolinium content was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) as 0.52 mmol / g. The Pm of the MRI polymeric contrast agent was determined by FPLC as 55 kDa with a polydispersity of 1.43. The T1 relaxivity of the conjugate was determined as 10.4 mM'1 s'1 per Gd3 + complexed using a B1 homogeneity-corrected Look-Locker technique in the 1.5V GE NV / Cvi scanner with the LX 8.4 operating system at temperature ambient (19 ° C). The chemical structure of the Mromo macromolecular contrast agent is shown in Figure 1.

P-Dex Synthesis

HPMA (1 g, 0.00698 mol), MA-GG-OH (0.156 g, 0.00078 mol), MA-FITC (0.02 g, 0.00004 mol) and AIBN (0.007 mg, 0,) were dissolved 000043 mol) in a mixture DMSO (1 mL) and MeOH (8 mL). The solution was transferred to a vial and purged with N2 for 5 minutes. Then it was polymerized at 50 ° C for 24 hours. The polymer was re-precipitated twice to produce approximately 1 g of copolymer. It was further activated with a large excess of hydroxy succinimide (HOSu) and then reacted with hydrazine. After the new precipitation, dexamethasone was conjugated with the copolymer in the presence of 1 drop of acetic acid in DMF. The conjugate was purified with an LH-20 column and lyophilized to obtain the final conjugate (structure shown in Figure 6) with a dexamethasone content of 49 mg / g (conjugate).

Adjuvant-induced arthritis rat model

Male Lewis rats (175-200 g) were obtained from Charles River Laboratories (Portage, MI) and allowed to acclimatize for at least one week. To induce arthritis, Mycobacterium tuberculosis H37Ra (1 mg) and LA (5 mg) were mixed in paraffin oil (100 pL), sonicated and injected s.c. at the base of the rat's tail (20). The rats were then randomized into 3 rats / group. The progression of the inflammation of the joint was followed by the measurement of the diameter of the ankle joint with a caliper. Special care was given to the rats as inflammation developed to ensure availability and access to water and food. The MRI contrast agents used for the study were injected directly into the jugular vein while the animal was anesthetized with Ketamine and Xylazine.

Visualization of the accumulation of albumine in plasma in joints with RA

Evans blue dye (EB, 10 mg / kg in saline solution) was injected in healthy rats and with AIA through the tail vein. Extravasation and accumulation of dye in the areas of inflammation of the joints can be observed visually as an aspect of the blue pigment. Photographs of the ankle and legs were taken before and 8 hours after the injection.

Histology

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At necropsy, the main organs and limbs were removed and fixed with 10% phosphate regulated formalin for 24 hours. The organs were then dehydrated and embedded in paraffin for routine histopathological analysis. The limbs were gradually dehydrated in ascending concentrations of ethanol and embedded in poly (methyl methacrylate). Sections of the entire joint, including uncalcified bone, were cut with a low speed saw using diamond blades. The sections were mounted on plastic slides, ground to approximately 50 pm thick and superficially tined using modified Giemsa tincture for plastic sections (21). The joints (knee, ankle, tarsus and metatarsals) of the same animals that were visualized by MRI were evaluated to determine the presence of inflammation and tissue damage using the histological sections. A Bio-quant histomorphomethane system was used to measure the surface of bone erosion.

Bone mineral density

Bone mineral density (BMD) of the bones in the artistic joints was measured by dual peripheral X-ray absorption (pDXA, Norland Medical Systems) adapted for small animals. For this, the intact hind limbs were used and the exploration region included the ankle and foot bones. The coefficient of variation between the measurements was less than 1%.

Visual and histological examination of rats with AIA

The development of adjuvant-induced arthritis in rats is well described in the literature (20), and is briefly summarized here. After injection of the adjuvant, the changes begin to become apparent approximately 9 days later. This includes some inflammation around the eyes and enlarged and soft external genitalia. Inflammation and swelling of the ankle joints of the front and back limbs become apparent about 12 days after the injection of the adjuvant.

At necropsy at 15 days after the injection of the adjuvant, the inflammation of the peritoneum (peritonitis) can be observed. Occasionally, inflammation of the gastrointestinal (GI) tract and fluid retention in the peritoneal cavity are also detectable. In general, the mayona of the vital organs appear to be normal, except that the spleen is usually enlarged with visual evidence of inflammation.

However, in the histopathological examination, all the organs examined showed signs of chronic inflammation. Testicular tissue shows inflammation in the membranes around the testicle, with small granulomas in the epididymis that are detected. The pericardial tissue demonstrates chronic inflammation, which could easily allow the accumulation of fluid in the pericardial tissue. The renal tissue includes multifocal areas of granuloma formation in the cortical tissue with some inflammation on the capsule, particularly along possible changes in the serous surface. Splenic tissue shows multifocal areas of necrosis surrounded by neutrophils and epithelioid cells. Plasma cells and lymphocytes are responding around this process, indicating a rather severe inflammatory response throughout the splenic tissue.

The histological images of the hind legs of the rats with AIA are presented in Figure 2. At a lower magnification, swelling of the region of the ankle joint and the legs of the rats with AIA is evident (Figure 2A). Extensive inflammation, synovitis, destruction of bone and cartilage is evident (Figures 2B to 2D). Inflammatory cells are observed around the larger vessels (Figure 2E). In contrast, the knee joints of rats with IAA are typically less affected by the inflammatory process (Figure 2F).

Visual examination of rats with AIA after Evans blue injection

For this experiment, the rats were injected EB 15 days after the injection of the adjuvant. At this time, there is a strong inflammatory reaction evident in the ankle joints. After the injection of EB, there was a gradual accumulation of blue color in the inflamed hind leg and front legs of the rats with AIA with high color density located around the tarsus and the carpus. Some deep blue spots were also observed in some digits of the leg. Photos taken before and after the injection of the EB dye confirm that the areas with dye accumulation correspond to those with marked inflammation. In healthy control rats that received EB, the dye was not located in the joint areas as observed in rats with AIA.

MR imaging

The imaging of rats with AIA with P-DOTA-Gd3 + as a contrast agent. Immediately before the injection of the contrast agent P-DOTA-Gd3 +, a baseline MRI was performed. The animals were then injected with the contrast agent and MRI scans were performed at different time intervals. The acquired images were subsequently processed using the maximum intensity projection algorithm (PMI). The PMI images resulting from the animals are represented chronologically in Figure 3.

As shown in the image of the base line (Figure 3A) before the contrast injection, the intestine and stomach of the animal are clearly visible probably due to fluid retention. There are also several irregular spots on the lower abdomen, which can be attributed to the injection site or sites i.p. of anesthetic agents. An area in the scrotum, adjacent to the testicles in the anatomical region of the epididymis and associated tissue,

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It also shows a diffuse MRI signal, perhaps due to its fatty content or accumulation of liquid. The bright spot in the right sciatic region may represent the retention of Kids in an inconsistent lymph node called 1c. The ischiatic foramen, which is also evident in some of the later images of this animal. No significant signal of Rm was observed in the hind limbs. However, the ankle joints clearly enlarged in the latent image when compared to similar images of the controls.

Five minutes after the injection of the macromolecular contrast agent, there was a substantial MRI signal in the kidneys (Figure 3B). A detailed examination of the two-dimensional images of a single plane indicates that at this time most of the contrast agent is in the renal cortex with little in the medulla. Due to the general increase in image contrast after injection, the bladder became apparent as a negative (dark) image as the oval-shaped structure in the lower left abdominal area. There is also an increase in contrast in the liver, spleen and bone marrow. The main blood vessels are clearly defined, while the smaller vessels are not as obvious, probably due to the limited image resolution (approximately 0.5 mm) with the 1.5 T MRI scanner. However, the vessels appear larger, perhaps dilated, than those observed in healthy controls. Except for some bone marrow absorption, little sign of significant contrast was evident at this time in the inflamed region of the ankle.

In the MR images (Figure 3C) of the rats with AIA acquired 1 hour after the injection, the signal in the kidney bark was significantly reduced compared to the previous time (5 minutes). However, now most of the contrast seems to be concentrated in the renal medulla and pelvis. Both ureters contain contrast material and a substantial signal is now evident in the urinary bladder. There seems to be a slight decrease in the RM contrast signal in the liver, spleen and blood vessel. Interestingly, several "hot spots" begin to appear around the tarsus, where the most severe inflammation occurs in this animal model.

From the MR images acquired 2 hours (Figure 3D) and 3 hours (Figure 3E) after the injection of the macromolecular contrast agent, a gradual reduction of the RM contrast signal in the kidney (cortex and spinal cord) was observed ), liver, spleen and vasculature. There was, however, an accumulation of contrast material in the urinary bladder. The "hot spots" detected around the tarsal in the 1-hour scan continue to expand and increase in contrast in the 2-hour (Figure 4-B) and 3-hour images.

When the rats were scanned again 8 hours after the injection, the acquired MR images (Figure 3F) show a very reduced MR signal in all vital organs and blood vessels with an essentially undetectable bladder, despite the body signal In general, it remains slightly larger than the base image. Surprisingly, however, the MR contrast signal is substantially increased in the ankle joint and the metatarsal region (Figure 4C). The initial "hot spots" disappear and the MR contrast signal is distributed more evenly around the joint tissue. There is also some contrast signal in the posterior joints of the knee, but with a much smaller intensity and size (Figure 4D).

Subsequently, the animals were scanned again at 32 hours and 48 hours after injection, respectively (Figures 3G and 3H). The overall contrast increase of the RM signal continued to decrease compared to what was observed in the 8-hour images. However, the decrease in image contrast in the ankle joint tissue seems to be much slower than that seen in other tissues and organs. Even after 48 hours, the potentiating effect of injected macromolecular contrast agents remains visible on the back ankle and leg tissue.

Image formation of healthy rats with P-DOTA-Gd3 + as a contrast agent. In the MR images (Figure 3J) taken 5 minutes after the injection of the macromolecular contrast agent, the kidneys of healthy animals showed an extremely strong contrast signal. Two-dimensional images on a single plane indicate that the MRI contrast resides in both the cortex and the medulla. The two lateral ureters are partially visible. The urinary bladder is filled with a significant amount of contrast medium. The liver, spleen and bone marrow were visible in the image when compared to the baseline image. The main blood vessels were also highlighted, including the abdominal aorta and the inferior vena cava. The arrangement and appearance of these vessels seems to be normal. No contrast signal was detected outside the large vessels in the hind legs.

In the MR images taken at 1 hour (Figure 3K) and at 2 hours (Figure 3L) after the injection, there is little contrast in the kidney and medullary cortex, but there is some contrast signal in the renal pelvis and the bladder The improvement in the contrast of the vasculature was slightly reduced compared to that observed 5 minutes after the injection. At 8 hours (Figure 3M) after injection, the contrast medium was completely removed from the urinary tract. At this time, some of the great vessels were still evident, although less than in previous moments. The images taken at 48 hours (Figure 3N) after the injection replicate the images of the base line without detectable contrast enhancement. As expected, all MRI images taken at different times after the injection did not show increased contrast in the joints of the animal's hind limbs.

Imaging of healthy rats and with AIA with OMNISCAN as contrast agent.

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The images acquired with the MR enhancement of a low molecular weight paramagnetic contrast agent OMNISCAN (bismethylamide gadolinium complex of diethylenetriamine pentaacetic acid) were obtained similarly to those injected with P-DOTA-Gd3 +.

From the sequence of images presented in Figure 3 (3O to 3X), a very rapid increase in tissue contrast was observed 5 minutes after the injection in both healthy rats and AIA (Figures 3O and 3T). However, the contrast improvement decreased rapidly, accompanied by rapid renal clearance of the contrast medium. At 8 hours (Figures 3Q and 3W), the improvement disappeared basically. Interestingly, the 5-minute images (Figure 3O) of the rats with AIA reveal a significant improvement in the contrast in the inflamed ankle joints, which had been removed in the 2-hour scan (Figure 3P). However, no such observation was found in healthy rats. Basically, no increase in the contrast of the blood vasculature was observed in all MR images enhanced with OMNISCAN.

As shown in Figure 3, all vital organs in rats with AIA showed greater absorption of P-DOTA-Gd3 + than healthy rats. In addition, the removal of the contrast agent in these organs was slower than in healthy rats, especially in the kidneys. These observations are consistent with the histological findings that all organs in rats with IAA, including heart, liver, lung, kidney and spleen fear some chronic granulomatous inflammation. The vasculature of these inflamed tissues is usually more porous, allowing greater extravasation of macromolecules to the interstitial tissue. These can lead to organ dysfunction, such as the delay in renal clearance of the polymeric contrast agent compared to healthy rats. However, the greatest elimination of P-DOTA-Gd3 + from these organs was completed in a few hours (<8 h) in rats with AIA. Compared to normal rats, the main blood vessels appear to be dilated in rats with IAA. This observation may be due to the level of prostaglandins overregulated in this model of systematic inflammation (26). It can also help explain the fastest polymer extravasation observed.

Interestingly, however, extravasation in inflamed ankle joints was delayed for a short time (1 ~ 2 h) in the AIA model (Figures 3A-3H). The "hot spots" of high RM contrast signal appeared later around the tarsus indicating high local concentrations of P-DOTA-Gd3 +. These "hot spots" also reveal the location of possible local damage in and around the joint. The polymer continues to be extravasated, diffusing, accumulating in the ankle joints and the highest concentrations were observed in the images 8 hours after the injection (MR images enlarged in a single plane, Figure 4). Because some increased concentrations of polymer were still observed in the joint 32 hours after injection, it seems that the removal of the polymer from the joint is relatively slow. By correlating the accumulation of polymers, as detected by MRI, with the histology of the same tissues (Figures 2A, 2B and 2D), it is evident that the accumulation of the polymer correlates with the degree of inflammation. As observed in the 8-hour MRI images, the accumulation of P-DOTA-Gd3 + in the knee joints was much smaller than that observed in the ankle joints (Figure 4D). This finding coincides very well with the amounts and severity of the inflammation observed histologically in the joints (Figure 2F). In contrast to the observation in rats with AIA, no extravasation of P-DOTA-Gd3 + was observed to the ankle or knee joints in healthy control rats.

The data suggest a pharmacokinetic profile with a renal elimination mechanism and a redistribution of the HPMA copolymer (marked with DOTA-Gd3 +) of the main organs and the blood circulation compartment in the arthritis inflammatory joints. Compared to the normal animal, the result of the MR images of the AIA model clearly demonstrates a very selective effect of addressing and accumulation of polymers in the artistic joints with a time interval of approximately 1 to 2 days after a single bolus injection . Since the majority of current anti-arthritis drugs may not specifically target the artificial joints and damaged tissues, together with low efficacy, the observed objective and the accumulation of polymeric delivery systems to the artistic joints demonstrate the great efficacy and numerous Potential applications of this invention for the administration and treatment of drugs, for example, of rheumatoid arthritis.

Likewise, imaging and evaluation of inflammatory tissues or organs, such as arthritis joints, with a MRI macromolecular contrast agent, also provides much better imaging results, as shown in Figures 3F and 4C, when it is compared with the low molecular weight MRI contrast agent, such as OMNISCAN (Figure 3O). The invention allows a longer timeframe for a longer and / or more detailed and / or sophisticated imaging process, which cannot be optimally performed, with current low molecular weight imaging agents, such as OMNISCAN. More anatomical details can be revealed with these imaging agents, which can have many applications, such as pre-thermal evaluation of the therapeutic effects of experimental antiarthritis drugs in an animal model and the clinical evaluation of the patient's response to treatment. Similar benefits can be obtained when the invention is used with PET, CT or gamma scintigrarta imaging agents. When MR, PET, CT or gamma scintigrarta imaging agents are conjugated with the polymeric delivery systems described in this invention, they will be able to provide powerful molecular imaging tools for the understanding of inflammatory diseases, such as rheumatoid arthritis.

Although the enhanced permeability of the vasculature in the artistic joints may be comparable to those found in the solid tumor, the retention of the polymer in the joint tissue may vary according to the stage of the

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disease. A rapid mechanism of drug excision is applied to ensure the effective release of the drug from the macromolecular carrier. A person with ordinary knowledge in the art will recognize that some pathological features of the artistic joints can be exploited for this. For example, drug release from the polymer can be facilitated by things such as very high extracellular enzymatic activities (for example, cathepsin K, MMP, etc.) (28), low pH, hypoxia or elevated temperature (29). Similarly, measures that increase the retention of extravasated polymers in the joints according to the invention (for example, polymeric drug conjugates) can also be used. The incorporation of targeting fractions, which bind to the negatively charged cartilage (30), to the surface of the newly eroded bone (21) or to the rheumatic factors enriched in the joints with RA, can also be used to increase the uptake and retention of the Polymer in the tissue of the joint. It is also believed that, by increasing the molecular weight of the polymeric carrier, greater retention of the polymer can be achieved in the AR joint. Antiartic drugs, such as glucocorticoids, can be used in the drug delivery system of the invention.

As will be recognized by one of ordinary skill in the art, anti-inflammatory drugs, anti-arthritis drugs, targeting fractions and imaging agents, as used herein, include acceptable salts, esters or salts of such esters. For example, glucocorticoids include pharmaceutically acceptable salts and esters thereof, therefore, when a drug is described, for example, dexamethasone, pharmaceutically acceptable salts thereof, such as dexamethasone palmitate, are also described.

The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: that is, salts that retain the desired biological activity of the original compound and do not impart unwanted toxicological effects thereto.

Base addition salts and pharmaceutically acceptable acid addition salts are known in the art (see, for example, Berge et al., "Pharmaceutical Salts", J. of Pharma Sci., 1977, 66, 1-19 ) REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition (1990, Mack Publishing Co., Easton, Pa.) AND GOODMAN AND GILMAN'S,

THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (10th edition, (2001)).

The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that becomes an active form (ie drug) within the body or cells thereof by the action of endogenous enzymes or other chemical products and / or conditions. .

In addition, the currently available protein drugs and the orally available low molecular weight drug can also benefit from the principles illustrated in the invention. For example, extravasation of the polymer injected into the joints with RA was delayed for 1 to 2 hours. Therefore, for protema or peptide drugs, they must survive this period of time against liver and kidney clearance. Protective or peprtic drugs can be stabilized by methods known in the art, for example, Protein PEGylation and / or modification of the polymer's main structure can provide a beneficial means to solve this problem (3).

Using modern MR imaging techniques, the specific accumulation of macromolecules in artistic joints was observed in the rat model of adjuvant-induced arthritis. There was an excellent correlation between the uptake and retention of the polymer marked with MRI contrast agent with histopathological features of inflammation and local tissue damage. The methodology used in this study showed that macromolecular imaging agents (polymeric delivery systems conjugated with imaging agents of MRI, CT, PET, gamma scintigrarta) are powerful imaging and evaluation tools for inflammatory diseases, such as rheumatoid arthritis. The use of macromolecular imaging agents also demonstrates the usefulness of the delivery system in order to address a drug, which is a beneficial improvement over current treatments, for example, for the treatment of rheumatoid arthritis. The invention provides the ability to increase the therapeutic potential and the dosage window of the drugs by reducing their side effects. In addition, the invention may have a longer half-life in blood circulation when compared to low molecular weight drugs, which may increase the bioavailability of the drug. In addition, the invention can be used to hydrophobicize a hydrophilic drug and, particularly for peptide-based drugs, reduce immunogenicity.

To demonstrate the superior therapeutic effects of the invention, an HPMA copolymer containing a targeting fraction was synthesized with an antiartic drug. Hydrazine was used as the targeting fraction, since it can be attached to negatively charged fractions in the cartilage. The antiarthetic drug, dexamethasone, was linked to the polymer's main chain (P-Dex) through a pH sensitive hydrazone bond as illustrated in Figure 6. The polymer with the bound hydrazine and dexamethasone was then injected into rats with AIA (4 / group) at day 13 after arthritis induction. A single dose of 10 mg (P-Dex) / kg was administered. As a control, the same dose of low molecular weight dexamethasone sodium phosphate (Dex) was divided into 4 equal doses and one dose was administered every day to another group of rats with IAA (4 / group) from day 13-16 after the induction of arthritis. As shown in Figure 7, both groups of animals showed a dramatic decrease in swelling of the ankle joint after injections on day 13. However, with the cessation of daily injections of the control Dex, the inflammation worsened. quickly while the inflammation in the P-Dex group had a prolonged suppression. These significant advantages of the treatment with P-Dex can be attributed to the specific addressing and improved retention (due to the fraction of addressing to the cartilage) of the polymeric delivery system to the

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Artificial joints of animals.

To reinforce the statistics of the superior therapeutic effects observed in the delivery system, a study was carried out with larger groups of animals (7 / group). One of the significant impacts of rheumatoid arthritis inflammation is bone damage in the joints, which is evident in Figure 8 of animals without treatment (saline solution). Glucocorticoids, such as dexamethasone (Dex), can delay bone erosion by reducing joint inflammation, as is evident in Figure 8 of animals treated with Dex. However, such improvement can be strongly reinforced if Dex is conjugated with an HPMA copolymer. The inflammation inhibition is prolonged (Figure 7) and the bone is well preserved in the group of animals treated with P-Dex with a BMD similar to the healthy group. A more dynamic factor to consider in bone metabolism is the degree of bone erosion. The eroded surface of the bone correlates directly with the recruitment and activity of the osteoclasts, which are the cells responsible for bone resorption and the development of bone damage. In Figure 9, the bone erosion surface data for all treatment groups is summarized. Again, the group with P-Dex showed a lower percentage of erosion surface compared to the group with Dex. Histological analysis of the artistic joints with different treatments also confirmed the superiority of the treatment with P-Dex (Fig. 10). The water soluble polymer main chain of the invention is an HPMA copolymer. The invention may optionally include one or more targeting fractions, which may be used to direct the delivery system to a specific tissue, such as bone, cartilage, etc. Illustrative examples of addressing fractions include, but are not limited to bisphosphonates, quaternary ammonium groups, peptides (e.g., oligo-Asp or oligo-Glu), aminosalidic acid and / or antibodies or fragments or derivatives thereof (e.g., Fab, humanized antibodies and / or scFv). An address fraction can be linked to the main polymer chain through covalent or physical bonds (links). Optionally, the spacers between an address fraction and the main polymer chain can be cleaved after a stimulus that includes, but is not limited to, changes in pH, presence of a specific enzymatic activity (e.g., cathepsin K, MmP, etc. .), changes in oxygen levels, etc. The spacers between the therapeutic agent and the main polymer chain can be cleaved by a spur that includes changes in pH. Optionally, a bioassay label (or labels) can be attached to the main polymer chain. It can be any label known in the art, including, but not limited to, a radioisotope, biotin, gold, etc. Its average mole percent per polymer chain can vary from 0% to about 50%.

The bioassay marker and / or the targeting fraction can be linked to the water soluble polymer main chain by means of a spacer. The spacers are known in the art and the person with ordinary skill in the art can select a spacer based on length, reactivity, flexibility and the like. For example, a spacer may be an alkyl or alkyne having one to 50, preferably one to 15 carbons. The spacer may be a peprtdica sequence (for example, selected from all natural amino acids) having one to 20, preferably one to 10 residues. In yet another example, a spacer may contain a hydrazone bond that is cleavable under an acidic pH. These spacers can be cleaved after a stimulus that includes, but is not limited to, changes in pH, presence of a specific enzymatic activity (for example, cathepsin K, MMP, etc.), changes in oxygen levels, etc.

Optionally, the biodegradable crosslinking shown in Figure 5 can crosslink, to some extent, the linear polymeric main chain. The resulting supply system still retains its solubility in water. The bond in sf is preferably cleavable under physiological conditions.

As one skilled in the art will appreciate, each class (eg, therapeutic agent, targeting fraction, bioassay marker, separator and / or imaging agent) can comprise any number of different compounds or compositions. For example, the therapeutic agent may consist of a mixture of one or more FAINE and one or more glucocorticoids, such as a combination of dexamethasone and hydrocortisone. Therefore, the invention provides the advantage that any combination of different therapeutic agents, targeting fractions, bioassay markers, spacers and / or imaging agents can be incorporated on the water soluble polymer main chain. As a result, a drug delivery or imaging system can be created with two or more different therapeutic agents and / or two or more different targeting fractions and / or two or more different bioassay markers, and / or two or more spacers different (one or more of which may be cleavable, where the splinter splinter may be different for different spacers) and / or two or more imaging agents. For example, one or more imaging agents can be combined with one or more therapeutic agents, to produce a combination of drug / imaging agent that, for example, can be used to treat and / or monitor the subject. An exemplary embodiment of such a drug / imaging agent is a method of determining the effects of a particular drug or combination of drugs. For example, the drug / imaging agent may contain a candidate drug in which the imaging agent allows improved monitoring of the effects of candidate drugs. In another exemplary embodiment, the drug / imaging agent can also be used to treat a subject and to monitor the response of the subjects to the treatment.

An effective amount of a drug is well known in the art and changes due to age, weight, severity of the condition of the subject, the particular compound in use, the concentration of the preparation and the mode of administration. The determination of an effective amount is preferably left to the prudence of a treating physician, but can be determined using methods well known in the art (37, 38). The compositions of the invention can be prepared using methods known in the art, for example, the preparation of a pharmaceutical composition is known in the art (37, 38).

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The compositions can be administered by any desirable and appropriate means. For in vivo delivery (ie, to a subject having arthritis or other inflammatory diseases), it is preferred that the delivery system be biocompatible and preferably biodegradable and non-immunogenic. In addition, it is desirable to provide a therapeutically effective amount of a compound in a physiologically acceptable vehicle. The injection in an individual can be subcutaneously, intravenously, intramuscularly, intraperitoneally, intraarticularly or, for example, directly in a localized area. Alternatively, in vivo delivery can be achieved through the use of a syrup, an elixir, a liquid, a tablet, a pfldora, a time-release capsule, an aerosol, a transdermal patch, an injection, a drip, an ointment , etc.

Abbreviations

AIA, adjuvant-induced arthritis; AIBN, 2,2'-azobisisobutyronitrile; APMA, N- (3-aminopropyl) methacrylamide hydrochloride; BMD, bone mineral density; COX-2, cyclooxygenase-2; CT, computed tomography; Dex, dexamethasone sodium phosphate; FARME, disease-modifying anti-rheumatic drugs; DIPEA, diisopropylethylamine; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra (acetic acid); DOTA-NHS ester, mono (N-hydroxysuccinimidyl ester) of 1,4,7,10-tetraazacyclododecane-1,4,7-tris (acetic acid) -10-acetic acid; DTPA-Gd3 +, gadolinium complex with pentaacetic diethylenetriamine acid; DXA, dual x-ray absorptometry; EB, Evans blue; PRM, permeability and enhanced retention; 18fDF-F, fluorodeoxyglucose; FITC, fluorescema isothiocyanate; FPLC, rapid liquid protein chromatography; HPMA, N- (2-hydroxypropyl) methacrylamide; ICP-OES, inductively coupled plasma optical emission spectroscopy; IL-IRa, interleukinal receptor antagonist; LA, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) propanediamine; MA-FITC, N-methacrylaminopropyl fluorescema thiourea; MA-GG-NHNH2, N-methacryloylglycylglycylhydrazine; Mn, number average molecular weight; MPA, mercaptopropionic acid; MRI magnetic resonance imaging; Pm, weighted average molecular weight; FAINE, symptomatic treatment with non-steroidal anti-inflammatory drugs; OA, osteoarthritis; OMNISCAN or gadodiamide is the injectable formulation of the gadolinium bismethylamide complex of pentaacetic diethylenetriamine acid; P-Dex, dexamethasone conjugate with HPMA copolymer, MA-GG-NHNH2 and MA-FITC through hydrazone binding (Figure 6); PET, positronic emission tomography; PHPMA, poly [N- (2-hydroxypropyl) methacrylamide]; poly (HPMA-co-APMA-co-MA-FITC), copolymer of HPMA, APMA and MA-FITC; P-DoTa, conjugation product of poly (HPMA-co-APMA-co-MA-FITC) and DOTA-NHS ester; P-DOTA-Gd3 +, purified complex of P-DOTA and Gd3 +; RA, rheumatoid arthritis; TA, room temperature; SEC, exclusion chromatography by size; scFv, single chain variable fragment; ATM, temporomandibular joint syndrome; TNF, tumor necrosis factor.

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Claims (42)

5 10 fifteen twenty 25 30 35 40 Four. Five 1. A pharmaceutical composition for use in the treatment of an inflammatory disease comprising: a water-soluble N-2 (2-hydroxypropyl) methacrylamide (HPMA) copolymer and an effective amount of an anti-inflammatory therapeutic agent bound to said water-soluble HPMA co-polymer, wherein the water-soluble HPMA copolymer is attached to the agent anti-inflammatory therapy through a spacer comprising a hydrazone bond. 2. The pharmaceutical composition for use according to claim 1, further comprising a targeting fraction attached to the water soluble HPMA copolymer. 3. The pharmaceutical composition for use according to claims 1 or 2, wherein the inflammatory disease comprises rheumatoid arthritis. 4. The pharmaceutical composition for use according to any one of claims 1-3, further comprising a bioassay label bound to the water soluble HPMA copolymer. 5. The pharmaceutical composition for use according to claim 1, wherein the anti-inflammatory therapeutic agent is a glucocorticoid. 6. The pharmaceutical composition for use according to claim 2, wherein the targeting fraction directs the composition to the bone or cartilage. 7. The pharmaceutical composition for use according to claim 2 or claim 6, wherein the targeting fraction is selected from the group consisting of bisphosphonates, quaternary ammonium groups, peptides, oligo-Asp, oligo-Glu, acid aminosalidlico, antibodies and fragments or derivatives in this. 8. The pharmaceutical composition for use according to claim 2, or 6-7, wherein the junction between the addressing fraction and the water soluble polymer is cleavable. 9. The pharmaceutical composition for use according to claim 2, or 6-7, wherein the bond between the addressing fraction and the water soluble polymer is non-cleavable. 10. The pharmaceutical composition for use according to claim 1, wherein the therapeutic agent is selected from the group consisting of proteins, peptides, FAINE, FARME, glucocorticoids, methotrexate, sulfasalazine, chloroquine, gold, gold salt, copper , copper salt, penicillamine, D-penicillamine, cyclosporine, and mixtures thereof. 11. The pharmaceutical composition for use according to claim 1 in an aqueous solvent or diluents for use in the delivery of an aqueous composition to a subject thought to have rheumatoid arthritis; Y allowing the accumulation and addressing of the pharmaceutical composition in an artistic joint, thus improving the treatment of arthritis. 12. The pharmaceutical composition for use according to claim 11, further comprising reducing a side effect of the therapeutic agent in tissues other than the arthritis joint. 13. The pharmaceutical composition for use according to claim 11, wherein the therapeutic agent is selected from the group consisting of FAINE, FARME, cyclooxygenase-2 inhibitor, a glucocorticoid, a tumor necrosis factor blocker and an antagonist of interleukin receiver 1. 14. A composition to imagine and evaluate an inflammatory disease comprising: a water soluble N- (2-hydroxypropyl) methacrylamide (HPMA) copolymer and an effective amount of a medical imaging agent bonded to said water soluble HPMA copolymer, and wherein said water soluble HPMA copolymer also comprises a anti-inflammatory therapeutic agent attached to said water soluble HPMA copolymer, wherein the water soluble HPMA copolymer is attached to the anti-inflammatory therapeutic agent through a spacer comprising a hydrazone bond. 15. The composition of claim 14, wherein the medical imaging agent is selected from the group consisting of at least one of an MRI, PET, CT and gamma scintigraphy agent. 16. The composition of claim 14, further comprising a targeting fraction attached to the water soluble HPMA copolymer. 17. The composition of claim 14, wherein the inflammatory disease comprises arthritis. 18. The composition of any of claims 14-17, further comprising a bioassay label bound to the water soluble HPMA copolymer. 5 10 fifteen twenty 25 30 35 40 Four. Five 19. The composition of any of claims 14-18, further comprising a spacer between the image-forming agent and the water-soluble HPMA copolymer, wherein the spacer is cleavable. 20. The composition of any of claims 14-18, further comprising a spacer between the imaging agent and the water soluble HPMA copolymer, wherein the spacer is not cleavable. 21. The composition of claim 14, wherein the addressing fraction directs the composition to a specific fabric. 22. The composition of claim 21, wherein the targeting fraction directs the composition to the bone or cartilage. 23. The composition of claim 21, wherein the targeting fraction is selected from the group consisting of bisphosphonates, quaternary ammonium groups, peptides, oligo-Asp, oligo-Glu, aminosalidic acid, antibodies and fragments or derivatives thereof. same. 24. The composition of claim 14, wherein the therapeutic agent is selected from the group consisting of proteins, peptides, FAINE, glucocorticoids, methotrexate, sulfasalazine, chloroquine, gold, gold salt, copper, copper salt, penicillamine, D-penicillamine, cyclosporine, and mixtures thereof. 25. A method to form the image of an inflammatory disease, the method comprising: Form the image of an inflammatory disease of a patient or an animal model with an MRI, PET, CT or gamma scintigraphy equipment before administering to said patient or animal model the composition of claim 14 and forming the image after having been administered to said patient or animal model the composition of claim 14. 26. The method according to claim 25, further comprising performing a biodistribution test. 27. The method according to claim 25 or 26, further comprising directing the water soluble HPMA copolymer to a specific tissue. 28. The method according to claim 27, wherein the addressing of the compound is directed to the bone or to the cartilage. 29. The method according to claim 27 or 28, wherein the addressing of the compound to a specific tissue comprises using a targeting fraction selected from the group consisting of bisphosphonates, quaternary ammonium groups, peptides, oligo-Asp, oligo -Glu, aminosalidic acid, antibodies and fragments or derivatives thereof. 30. The method according to any of claims 27-29, further comprising cleaving a link between the targeting fraction and the water soluble HPMA copolymer. 31. The method according to claim 27, wherein the imaging of an inflammatory disease of a patient or animal model improves with the compound comprising imaging of an artistic joint. 32. The method according to claim 25, wherein the medical imaging agent is selected from the group consisting of at least one MRI, PET, CT, gamma scintigraphy and combinations thereof. 33. The pharmaceutical composition for use according to claim 1, wherein the therapeutic agent comprises a plurality of different therapeutic agents. 34. The pharmaceutical composition for use according to claims 2, 6, or 7, wherein the addressing fraction comprises a plurality of different targeting fractions. 35. The pharmaceutical composition for use according to claim 34, wherein the plurality of different targeting fractions is directed to a plurality of tissues. 36. The pharmaceutical composition for use according to claim 4, wherein the bioassay marker comprises a plurality of different bioassay markers. 37. The pharmaceutical composition for use according to claim 1, wherein the spacer comprises a plurality of chemically distinct spacers. 38. The composition of claim 14, wherein the imaging agent comprises a plurality of distinct imaging agents. 39. The method according to claim 32, wherein the imaging agent comprises at least two imaging agents, wherein each of the two imaging agents is used in a different imaging technique. 40. A composition comprising a water soluble N- (2-hydroxypropyl) methacrylamide copolymer linked to a targeting fraction and a glucocorticoid through a pH-sensitive hydrazone bond. 41. The composition of claim 40, wherein the glucocorticoid is dexamethasone. 42. The composition of claim 40, wherein the address fraction is hydrazine. 5 43. The pharmaceutical composition for use according to claim 2, wherein the addressing fraction It is hydrazine.